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Abstract:

A scanning microscope includes a light source, illumination optics, and a
scanning device for moving the illumination focus across a target region
and doing so by varying the direction of incidence in which the
illuminating beam enters an entrance pupil of the illumination optics. To
incline the illumination focus relative to the optical axis of the
optics, the scanning device directs the illuminating beam onto a portion
of the entrance pupil that is offset from the center of the pupil and, in
order to move the illumination focus across the target region, the
scanning device varies the direction of incidence of the illuminating
beam within said portion. An observation objective is provided which is
spatially separated from the illumination optics and disposed such that
its optical axis (O3) is substantially perpendicular to the target region
and at an acute angle (α) to the optical axis (O1) of the
illumination optics.

Claims:

1. A scanning microscope (100, 200, 300, 400, 500); comprising: a light
source (20, 61, 64) for emitting an illuminating light beam (12),
illumination optics (10) for producing an elongated illumination focus
(16, 84) in an object (36) to be imaged, and a scanning device (33) for
moving the illumination focus (16, 84) across a target region to be
illuminated of the object (36) to be imaged and doing so by varying the
direction of incidence in which the illuminating light beam (12) enters
an entrance pupil (14) of the illumination optics (10), wherein in order
to incline the illumination focus (16, 84) relative to the optical axis
(O1) of the illumination optics (10), the scanning device (33) directs
the illuminating light beam (12) onto a portion of the entrance pupil
(14) of the illumination optics (10) that is offset from the center of
the pupil and, in order to move the illumination focus (16, 84) across
the target region to be illuminated, the scanning device varies the
direction of incidence of the illuminating light beam (12) within said
portion; and an observation objective (38) is provided which is spatially
separated from the illumination optics (10) and disposed such that its
optical axis (O3) is substantially perpendicular to the illuminated
target region and at an acute angle (α) to the optical axis (O1) of
the illumination optics (10).

2. The scanning microscope (100, 200, 300, 400, 500) as recited in claim
1, wherein in order to produce a light sheet (18) which is formed by the
moving illumination focus (16, 84) and is inclined relative to the
optical axis (O1) of the illumination optics (10), the scanning device
(33) varies the direction of incidence of the illuminating light beam
(12) in a plane of incidence which is parallelly offset from the optical
axis (O1) of the illumination optics (10); and the observation objective
(38) is disposed such that its optical axis (O3) is perpendicular to the
light sheet (18).

3. The scanning microscope (100, 200, 300, 400, 500) as recited in claim
2, wherein the scanning device (33) includes a control unit (32) and a
first adjustment unit (30, 50, 52, 60) coupled to the control unit (32)
for varying the inclination of the light sheet (18), as well as a second
adjustment unit (47) coupled to the control unit (32) for moving the
observation objective (38); and the control unit (32) controls the two
adjustment units (30, 50, 52, 60; 47) in a synchronized manner such that
the optical axis (O3) of the observation objective (38) remains oriented
perpendicular to the light sheet (18) adjusted by the first adjustment
unit (30, 50, 52, 60).

4. The scanning device (100) as recited in claim 3, wherein the
illumination optics (10) is mounted so as to be movable along an
adjustment direction (A) perpendicular to its optical axis (10); and the
first adjustment unit includes an actuator (30) which moves the
illumination optics (10) along the adjustment direction (A) to vary the
inclination of the light sheet (18).

5. The scanning device (200, 300) as recited in claim 3, wherein the
first adjustment unit includes: an optical displacement element (50, 60)
which is disposed in the path of the illuminating light beam (12) between
the light source (20) and the illumination optics (10) and is mounted so
as to be movable along an adjustment direction (A, C, D) transverse to
the optical axis (O1) of the illumination optics (10) and which displaces
the plane of incidence of the illuminating light beam (12) parallel to
the optical axis (O1) of the illumination optics (10); and an actuator
(30) which moves the displacement element (50, 60) along the adjustment
direction (A, C, D) to vary the inclination of the light sheet (18).

7. The scanning microscope (400) as recited in claim 2, wherein the first
adjustment unit includes: an aperture (52) which is disposed in the
region of the entrance pupil (14) of the illuminating optics (10) and has
an aperture opening (54) allowing a portion of the illuminating light
beam (12) to pass therethrough, and which is mounted so as to be movable
along an adjustment direction (A) perpendicular to the optical axis (O1)
of the illumination optics (10); and an actuator (30) which moves the
aperture (52) along the adjustment direction (A) to vary the inclination
of the light sheet (18).

8. The scanning microscope (100, 200, 300, 400, 500) as recited in claim
3, characterized by a photodetector (42) which, together with the
observation objective (38), forms a detection unit (46) which can be
moved by the second adjustment unit (47).

9. The scanning microscope (100, 200, 300, 400, 500) as recited in claim
1, wherein the scanning device (33) has a mirror system (24) which
reflects the illuminating light beam (12) emitted by the light source
(20, 61, 64) onto the portion of the entrance pupil (14) that is offset
from the center of the pupil and, and a mirror actuator which allows the
mirror system to be moved in order to vary the direction of incidence of
the illuminating light beam (12).

10. The scanning microscope (100, 200, 300, 400, 500) as recited in claim
2, wherein the portion of the entrance pupil (14) of the illumination
optics (10) that is traversed by the illuminating light beam (12)
occupies about 0.1% to 50% of the total area of the entrance pupil (14);
and the parallel displacement of the plane of incidence with respect to
the optical axis (O1) of the illumination optics (10) is about 4 to 96%
of the radius of the entrance pupil (14).

11. The scanning microscope (500) as recited in claim 1, wherein the
illuminating light beam (12) is composed of an excitation beam (62) and a
depletion beam (66) which are superimposed on each other before entering
the scanning device (33); and the illumination optics (10) produces an
excitation focus (80) from the excitation beam (62) and a depletion focus
(82) from the depletion beam (66), said excitation focus and said
depletion focus being superimposed on each other to form the illumination
focus (84).

12. The scanning device (500) as recited in claim 11, wherein the
depletion focus (82) has a spatial light intensity distribution which
exhibits a minimum in a plane (86) in which the illumination focus (84)
composed of the excitation focus (80) and the depletion focus (82) is
moved to generate a light sheet, as well as a maximum on both sides of
said plane (86).

13. A method for light-microscopic imaging of an object (36), comprising
the following steps: emitting an illuminating light beam (12), producing
an elongated illumination focus (16, 84) in the object (36) to be imaged
by means of illumination optics (10), moving the illumination focus (16,
84) across a target region to be illuminated of the object (36) and doing
so by varying the direction of incidence in which the illuminating light
beam (12) enters an entrance pupil (14) of the illumination optics (10),
wherein in order to incline the illumination focus (16, 84) relative to
the optical axis (O1) of the illumination optics (10), the illuminating
light beam (12) is directed onto a portion of the entrance pupil (14)
that is offset from the center of the pupil and, in order to move the
illumination focus (16, 84) across the target region to be illuminated,
the direction of incidence of the illuminating light beam (12) is varied
within said portion; and the target region illuminated with the inclined
illumination focus (16, 84) is imaged by an observation objective (38)
which is spatially separated from the illumination optics (10) and
disposed such that its optical axis (O3) is substantially perpendicular
to the illuminated target region and at an acute angle (α) to the
optical axis (O1) of the illumination optics 10).

14. The method as recited in claim 13, wherein in order to produce a
light sheet (18) which is formed by the moving illumination focus (16,
84), the direction of incidence of the illuminating light beam (12) is
varied in a plane of incidence which is parallelly offset from the
optical axis (O1) of the illumination optics (10); and the optical axis
(O3) of the observation objective (38) is disposed perpendicular to the
light sheet (18).

15. The method as recited in claim 14, wherein the inclination of the
light sheet (18) varies, and the observation objective (38) is moved in
synchronization with this variation in the inclination of the light sheet
(18) such that the observation objective (38) remains oriented with its
optical axis (O3) perpendicular to the adjusted light sheet (18).

16. The method as recited in claim 15, wherein the observation objective
(38) is moved together with a photodetector (42).

17. The method as recited in claim 14, wherein the illuminating light
beam (12) is composed of an excitation beam (62) and a depletion beam
(66); and an excitation focus (80) is produced from the excitation beam
(62) and a depletion focus (82) is produced from the depletion beam (66),
said excitation focus and said depletion focus being superimposed on each
other to form the illumination focus (84).

18. The method as recited in claim 17, wherein the spatial intensity
distribution of the depletion focus (82) is adjusted such that it
exhibits a minimum in a plane (86) in which the illumination focus (84)
composed of the excitation focus (80) and the depletion focus (82) is
moved to generate a light sheet, as well as a maximum on both sides of
said plane (86).

Description:

RELATED APPLICATIONS

[0001] This application claims priority to German Patent Application No.
102011051042.7 filed on Jun. 14, 2011, which is incorporated herein by
reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a scanning microscope having a
light source for emitting an illuminating light beam, illumination optics
for producing an elongated illumination focus in an object to be imaged,
and a scanning device for moving the illumination focus across a target
region to be illuminated of the object to be imaged and doing so by
varying the direction of incidence in which the illuminating light beam
enters an entrance pupil of the illumination optics.

BACKGROUND OF THE INVENTION

[0003] In the prior art, scanning microscopes have been proposed which are
used, for example, in fluorescence microscopy to excite dyes by laser
light to emit fluorescent radiation, which is then captured by a detector
to form an image of the object to be examined. The microscopes used in
this area of microscopy are, in particular, scanning confocal microscopes
which, unlike standard microscopes, do not illuminate the entire object
at a particular point in time, but produce a typically
diffraction-limited spot of light with which the object is scanned point
by point. The light signals detected by the detector at the individual
object points are then combined to form a complete image of the object.

[0004] Such a confocal microscope typically includes a scanning device of
the type called a point scanner, which directs the illuminating light
beam emitted by the light source into the entrance pupil of the
illumination optics. The illumination optics transform the illuminating
light beam entering its entrance pupil into a focused light distribution,
which will hereinafter be referred to as "illumination focus". The shape
and size of the illumination focus depend on the optical properties,
particularly the numerical aperture of the illumination optics. If the
illuminating light beam enters the entrance pupil of the illumination
optics centrally and perpendicularly; i.e., along the optical axis, then
the illumination optics produce an elongated illumination focus which has
a smaller extent transverse to the optical axis than along the optical
axis. Then, in order to scan the object, the illumination focus is moved
transversely to the optical axis by the point scanner varying the angle
of incidence at which the illuminating light beam enters the entrance
pupil of the illumination optics. This can be accomplished using, for
example, a movable mirror system.

[0005] Thus, in order to form a three-dimensional image using a confocal
microscope, the object must be scanned point by point in the manner
described above. Since this is a relatively complex process, a microscopy
technique referred to in the literature as selective plane illumination
microscopy (SPIM) was proposed recently. This technique uses an
illumination objective and an observation objective, which are arranged
at an angle of 90 degrees relative to each other. The illumination
objective, in cooperation with a cylindrical lens located upstream
thereof, produces an approximately two-dimensional distribution of
illumination light, which passes through the object along the optical
axis of the illumination objective. Such a light distribution is
frequently also referred to as "light sheet" or "light disk". The target
region of the object that is illuminated by this light sheet is imaged by
the observation objective onto a detection surface, such as a CCD, the
optical axis of the observation objective being perpendicular to the
light sheet. If the object is then moved through the light sheet, it is
possible to acquire tomographic images of the object using this
configuration. In order to produce as thin a light sheet as possible, the
illumination objective must have a correspondingly high numerical
aperture. Moreover, the free working distance of the illumination
objective must be large enough to prevent collision with the observation
objective. Accordingly, the numerical aperture of the illumination
objective determines the thickness of the light sheet, and thus, the
optical resolution along the optical axis of the observation objective.

[0006] In a modified SPIM method described in WO 2010/012980 A1,
illumination and detection are performed by one and the same objective.
To this end, the entrance pupil of the objective is under-illuminated at
an off-center position; i.e., the illuminating light beam passes through
a portion of the entrance pupil that is transversely offset from the
optical axis. A cylindrical lens upstream of the objective produces an
illuminating light sheet in the object, which light sheet is inclined to
the optical axis of the objective. The target region illuminated by this
light sheet is then imaged by the objective.

[0007] All of the above-described systems use a cylindrical lens to obtain
the desired oblique illumination of the object. However, the use of such
a cylindrical lens has disadvantages. For example, these devices are
designed exclusively for oblique illumination by means of a light sheet
and do not allow for a different use, such as point-by-point confocal
scanning. Moreover, it would be desirable to be able to vary the spatial
distribution of the light intensity of the light sheet produced for
oblique illumination. This is not possible using a cylindrical lens.

[0009] In methods using an illumination objective and a separate
observation objective, the two objectives are typically arranged at an
angle of 90 degrees relative to each other. This arrangement of the
objectives, where the illumination axis and the observation axis are
perpendicular to each other, can be a disadvantage in the imaging of
certain biological objects. For example, it is often not possible to
place spherical objects in a collision-free position under such a
right-angled arrangement of objectives. Such spherical objects are used,
for example, when tissue cultures in the eye of a mouse or rat are to be
examined. In such situations, the conventional arrangement of objectives
frequently results in shading by surrounding tissue.

[0010] It is an object of the present invention to improve a scanning
microscope of the type mentioned at the outset in such a way that it
allows collision-free light-microscopic imaging, even under difficult
geometric conditions.

[0011] According to the invention, this object is achieved in that, in
order to incline the illumination focus relative to the optical axis of
the illumination optics, the scanning device directs the illuminating
light beam onto a portion of the entrance pupil of the illumination
optics that is offset from the center of the pupil and, in order to move
the illumination focus across the target region to be illuminated, the
scanning device varies the direction of incidence of the illuminating
light beam within said portion, and in that an observation objective is
provided which is spatially separated from the illumination optics and
disposed such that its optical axis is substantially perpendicular to the
illuminated target region and at an acute angle to the optical axis of
the illumination optics.

[0012] The present invention proposes, first of all, to under-illuminate
the entrance pupil of the illumination optics with the illuminating light
beam at an off-center position by directing the illuminating light beam
onto a portion of the entrance pupil that is offset from the center of
the pupil. Underillumination of the entrance pupil; i.e., the feature of
not allowing the illuminating light beam to pass through the entire area
of the entrance pupil, and thus, not using the full aperture, produces a
widening of the (already elongated) illumination focus in both the
longitudinal and transverse directions. Since, in addition, the
illuminating light beam strikes the entrance pupil off-center, the
illumination focus is inclined relative to the optical axis of the
illumination optics.

[0013] The illumination focus widened and inclined in this manner can then
be used to sequentially generate a light sheet illuminating the target
region. This is accomplished by means of the scanning device, which
produces a suitable scanning motion of the illuminating light beam in the
entrance pupil of the illumination optics. This scanning motion
corresponds to a tilting of the illuminating light beam about a tilt
point located in the region of the entrance pupil. This means that the
illuminating light beam, which, of course, is not a ray in a mathematical
sense, but rather a bundle of light rays, remains (at least
approximately) stationary in the region of the entrance pupil, while at a
distance from the entrance pupil (toward the scanning device), it
performs, as it were, a pivoting motion relative to a reference direction
parallel to the optical axis. The tilting or pivoting motion of the
illuminating light beam is translated by the illumination optics into a
corresponding motion of the inclined illumination focus transverse to the
optical axis. The actual magnitude of the motion of the illumination
focus in the object depends on the specific design of the illumination
optics. However, what is essential is that the tilting of the
illuminating light beam caused by the scanning device is used in
accordance with the present invention to move the illumination focus
within the target region of the object and to thereby, as it were,
generate a light sheet which illuminates the target region.

[0014] Unlike the prior art approaches which employ a cylindrical lens to
produce a light sheet, the scanning microscope of the present invention
generates the light sheet sequentially by means of the scanning device,
which does so by moving the illumination focus across the target region
within one scanning period. To this end, the scanning period with which
the scanning device is operated may be adjusted to be significantly
shorter than the detection cycle of a photodetector onto which the target
region is imaged by the illumination optics. Accordingly, the
photodetector, which may, for example, be an area detector, CCD, CMOS,
APD array, or the like, "sees" the moving illumination focus in a
temporally and, therefore, spatially unresolved manner. Rather, it sees a
continuous light distribution in the form of a light sheet.

[0015] Since a scanning device which moves the illuminating light beam is
provided in a conventional scanning confocal microscope anyway, the
illuminating light sheet can be generated particularly efficiently using
the present invention. In particular, it is possible to use the same
microscope configuration for different applications. Thus, in order to
implement the application of the present invention, where the object is
obliquely illuminated by means of a light sheet, it is only necessary to
ensure off-center underillumination of the entrance pupil of the
illumination optics in a scanning confocal microscope having a per se
standard configuration. This can be accomplished, for example, by an
optical element inserted into the path of the illuminating light beam. If
the scanning microscope is to be subsequently operated with
point-by-point illumination again, then it is only necessary to remove
the optical element from the path of the illuminating light beam.

[0016] The scanning microscope of the present invention makes it possible
to acquire high-resolution cross-sectional images of the object. To this
end, the object is scanned step-by-step with the light sheet. This
scanning process may be carried out, for example, by moving the object
relative to the optical axis along or transverse to the optical axis.

[0017] In accordance with the present invention, an observation objective
is provided which is spatially separated from the illumination optics and
whose optical axis is substantially perpendicular to the light sheet
generated by the moving illumination focus and at the same time at an
acute angle to the optical axis of the illumination optics. An acute
angle is understood to be an angle less than the 90-degree angle provided
in conventional arrangements. Thus, this acute angle specifies the angle
between the illumination axis defined by the optical axis of the
illumination optics and the observation axis defined by the optical axis
of the observation objective. The magnitude of this angle is to be
selected depending on the specific configuration used, in particular
depending on the shape of the object to be examined. Preferably, the
acute angle is in a range from 20 to 80 degrees, a particularly preferred
value within this range being 50 degrees.

[0018] The scanning illumination focus which, according to the present
invention, is inclined defines an inclined object plane, which is imaged
by the observation objective. The observation objective is aligned such
its optical axis is perpendicular to said object plane. The present
invention allows the object plane to be aligned particularly easily by
displacing the illumination beam, according to the desired orientation,
from the center of the pupil at the entrance to the illumination optics.
The greater the displacement of the illumination beam from the center of
the pupil, the greater the inclination of the illumination focus, and
thus of the object plane defined by the moving illumination focus
relative to the optical axis of the illumination optics.

[0019] Preferred objective systems for use as the illumination optics and
the observation objective are those having a relatively large free
working distance (e.g., greater than 1 mm). Both illumination and
observation may be carried out using either a dry objective or an
immersion objective using water or another liquid as the immersion
medium.

[0020] Preferably, in order to produce a light sheet which is formed by
the moving illumination focus and is inclined relative to the optical
axis of the illumination optics, the scanning device varies the direction
of incidence of the illuminating light beam in a plane of incidence which
is parallelly offset from the optical axis of the illumination optics.
The observation objective is disposed such that its optical axis is
perpendicular to the generated light sheet. Assuming that the entrance
pupil of the illumination optics is circular in shape, the aforementioned
plane of incidence, when viewed from above, forms a straight line which
intersects the circle defined by the edge of the pupil in two different
points in the manner of a secant without crossing the center of this
circle. In the above-defined view from above, the illuminating light beam
then performs a tilting motion along this secant. The illumination optics
translate this tilting motion into a corresponding motion of the
illumination focus along a straight line running parallel to the
aforementioned secant. In this way, the desired light sheet for
illuminating the target region can be generated in a simple manner.

[0021] In another advantageous embodiment, the scanning device includes a
control unit and a first adjustment unit coupled to the control unit for
varying the inclination of the light sheet, as well as a second
adjustment unit coupled to the control unit for moving the observation
objective. The control unit controls the two adjustment units in a
synchronized manner such that the optical axis of the observation
objective remains oriented perpendicular to the light sheet adjusted by
the first adjustment unit. This design makes it possible to vary the
inclination of the light sheet, and thus of the object plane defined by
the light sheet. In addition, by suitably controlling the two adjustment
units, the control unit ensures that the observation objective is
adjusted in position to maintain the observation objective in
perpendicular alignment with respect to the object plane.

[0022] In one possible embodiment, the illumination optics are mounted so
as to be movable along an adjustment direction perpendicular to the
optical axis thereof. In this embodiment, moreover, the first adjustment
unit includes an actuator which moves the illumination optics along the
adjustment direction to vary the inclination of the light sheet. By
moving the illumination optics perpendicularly to its optical axis, the
portion of the entrance pupil through which the illuminating light beam
enters can be moved away from or toward the center of the pupil to
increase or reduce the inclination of the illumination focus.

[0023] In an alternative embodiment, the first adjustment unit includes an
optical displacement element which is disposed in the path of the
illuminating light beam between the light source and the illumination
optics and is mounted so as to be movable along an adjustment direction
transverse to the optical axis of the illumination optics and which
displaces the plane of incidence of the illuminating light beam parallel
to the optical axis of the illumination optics, the first adjustment unit
further including an actuator which moves the displacement element along
the adjustment direction to vary the inclination of the light sheet. The
actuator may move the displacement element either rectilinearly; i.e.,
perpendicularly to the optical axis of the illumination optics, or
alternatively along a curved path; e.g., pivoted about a pivot axis.
Furthermore, the actuator may be designed to allow the displacement
element to be completely removed from the path of the illuminating light
beam. In this case, the scanning microscope of the present invention may
be used like a conventional scanning confocal microscope where the object
is illuminated point by point.

[0024] The optical displacement element may, for example, be a transparent
plane-parallel or wedge-shaped plate which produces the desired
displacement of the illuminating light beam from the center of the
entrance pupil of the illumination optics.

[0025] In another alternative embodiment, the first adjustment unit
includes an aperture which is disposed in the region of the entrance
pupil of the illuminating optics and has an aperture opening allowing a
portion of the illuminating light beam to pass therethrough, and which is
mounted so as to be movable along an adjustment direction perpendicular
to the optical axis of the illumination optics, the first adjustment unit
further including an actuator which moves the aperture along the
adjustment direction to vary the inclination of the light sheet. The size
of the aperture opening determines the effective diameter of the
illuminating light beam entering the illumination optics. Preferably, the
size of the aperture opening is variable, so that the dimensions of the
illumination focus can be varied by adjusting the aperture opening. The
smaller the aperture opening, the greater the widening of the
illumination focus.

[0026] The off-center underillumination of the entrance pupil of the
illumination optics, as proposed by the present invention, can also be
implemented in a different way than described above. For example, it is
conceivable to displace the illuminating light beam parallel to the
optical axis of the illumination optics already in its path upstream of
the scanning device and to reduce its diameter according to the desired
underillumination. The parallel displacement of the illuminating light
beam may be accomplished, for example, by inserting glass plates in an
inclined orientation. If the scanning device includes a movable minor
system, such as is common in conventional scanning confocal microscopes,
the aforementioned parallel displacement may serve to cause the
illuminating light beam to strike the mirror system at a reflection point
which is offset from a reflection point at which the illuminating light
beam strikes the mirror system in a conventional confocal application.
This displacement, which is already implemented in the scanning device,
then results in the desired off-center position of the illuminating light
beam in the entrance pupil of the illumination optics.

[0027] Preferably, the scanning microscope of the present invention
includes an image sensor which, together with the observation objective,
forms a detection unit which can be moved by the second adjustment unit.
In particular, in this embodiment, the image sensor and the observation
objective are jointly adjusted in position to at all times maintain the
image sensor and the observation objective aligned with respect to each
other.

[0028] In a particularly preferred embodiment, the illuminating light beam
is composed of an excitation beam and a depletion beam which are
superimposed on each other before entering the scanning device. The
illumination optics produces an excitation focus from the excitation beam
and a depletion focus from the depletion beam, the excitation beam and
the depletion beam being superimposed on each other to form the
illumination focus. In fluorescence microscopy, such a depletion beam may
be used, for example according to the so-called STED method, to increase
the spatial resolution of the light-microscopic image beyond the
diffraction limit. In the STED method, fluorescent dyes, which are used
for labeling individual regions of the object, are selectively depleted
by the depletion beam in a manner known per se, and thus, as it were,
switched off in order to increase the resolving power. In the scanning
microscope of the present invention, the use of the STED method makes it
possible to narrow the effective width of the illumination focus, and
thereby make the resulting light sheet thinner, in order to increase the
resolution, said narrowing being achieved by superimposing the excitation
beam with the depletion beam. Since the depletion beam is superimposed on
the excitation beam before reaching the scanning device, the two
superimposed light beams are jointly tilted by the scanning device in the
entrance pupil of the illumination optics.

[0029] Preferably, the depletion focus has a spatial light intensity
distribution which exhibits a minimum in a plane in which the
illumination focus composed of the excitation focus and the depletion
focus is moved to generate a light sheet, as well as a maximum on both
sides of said plane. While in conventional STED applications, the
depletion focus is donut-shaped in cross section, the aforementioned
embodiment provides a focus which in cross section has two intensity
maxima (above and below the plane in which the illumination focus is
moved to generate the light sheet) and a minimum between said maxima.
Preferably, this cross-sectional intensity profile is symmetric and
exhibits two maxima of equal magnitude and a zero crossing as a minimum
therebetween.

[0030] This advantageous embodiment takes advantage of the fact that STED
depletion is not necessary, and even unwanted, in the plane in which the
illumination focus is moved to generate the light sheet. In the approach
of the present invention, the intention is to generate a light sheet that
has as large a surface area as possible and, at the same time, is as thin
as possible to be able to acquire cross-sectional images with high
spatial resolution. The area of the light sheet is determined by the
length of the moving illumination focus, while the thickness of the sheet
is determined by the extent of the illumination focus transverse to the
plane in which the illumination focus is moved. The aforedescribed
depletion focus is specifically shaped to reduce the excitation effect of
the excitation focus only in the direction of the transverse extent of
the focus, but not in the direction of its longitudinal extent.

[0031] It is conceivable to equip the scanning microscope of the present
invention with an element serving to vary the intensity of the
illuminating light beam within a scanning period during which the
illumination focus is moved across the target region. This embodiment
uses the fact that the light sheet illuminating the target region is
sequentially generated by the moving illumination focus. This makes it
possible to achieve modulated or patterned illumination of the target
region. To this end, the intensity of the illuminating light beam is
varied within the scanning period during which the illumination focus
scans across the target region. In this way, the intensity of the
illuminating light beam can be adjusted as desired at any point of time
within the scanning period, and thus, at any position of the target
region. The element used for varying the intensity of the illuminating
light beam within the scanning period may be, for example, an
acousto-optic tunable filter (AOTF), an acousto-optic modulator (AOM), or
an electro-optic modulator (EOM).

[0032] According to another aspect of the present invention, there is
provided a method for light-microscopic imaging of an object having the
features of claim 13.

[0033] The method of the present invention can be advantageously used in
localization microscopy. Using controlled illumination, it is possible to
achieve single-molecule detection. The locations of the fluorescent dyes
used can then be identified by determining the centroids of the detected
photons.

[0034] The method of the present invention may be particularly
beneficially used for imaging spherical biological objects such as, for
example, a tissue culture in the eye of a mouse or rat.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will now be described in greater detail with
reference to the drawings, in which:

[0036]FIG. 1a is a schematic view wherein the entrance pupil of
illumination optics for producing an illumination focus is fully
illuminated, as is the practice in conventional scanning confocal
microscopy;

[0037]FIG. 1b is a top view of the illumination focus shown in FIG. 1a;

[0038]FIG. 2a is a schematic view showing how scanning is performed by
tilting an illuminating light beam entering the entrance pupil of the
illumination optics, as is the practice in conventional scanning confocal
microscopy;

[0039]FIG. 2b is a top view of the illumination focus shown in FIG. 2a;

[0040]FIG. 3a is a schematic view illustrating the underillumination of
the entrance pupil of the illumination optics with the illuminating light
beam, as proposed by the present invention;

[0041]FIG. 3b is a top view of the illumination focus shown in FIG. 3a;

[0042]FIG. 4a is a schematic view showing how scanning is performed
according to the present invention by tilting the illuminating light beam
that under-illuminates the entrance pupil of the illumination optics;

[0043]FIG. 4b is a schematic view illustrating a motion sequence of the
illumination focus shown in FIG. 4a;

[0044] FIG. 5 is a view of a scanning confocal microscope according to a
first embodiment;

[0045]FIG. 6 is a schematic view showing how the illumination focus is
inclined by moving the illumination optics as depicted in FIG. 5;

[0046] FIG. 7 is a schematic view illustrating the alignment of the
observation objective to the inclined illumination focus;

[0047]FIG. 8 is a view of a scanning confocal microscope according to a
second embodiment;

[0048]FIG. 9 is a view of a scanning confocal microscope according to a
third embodiment;

[0049]FIG. 10 is a view of a scanning confocal microscope according to a
fourth embodiment;

[0050]FIG. 11 is a schematic view showing how the illumination focus is
inclined by moving an aperture plate provided in the scanning microscope
shown in FIG. 10;

[0051]FIG. 12 is a view of a scanning confocal microscope according to a
fifth embodiment, which operates according to the STED method;

[0052]FIG. 13a is a schematic view illustrating the superposition of an
excitation focus with a depletion focus; and

[0053]FIG. 13b is a top view of the excitation focus and the depletion
focus shown in FIG. 13a.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The principle of off-center underillumination of the [entrance]
pupil according to the present invention will now be described with
reference to FIGS. 1 through 4. In particular, it will be described how,
according to the present invention, illumination optics 10 produce an
illumination focus 16 from an illuminating light beam 12 incident on an
entrance pupil of illumination optics 10. In this connection, it should
be noted that the above-mentioned figures are purely schematic and are
merely intended to facilitate the understanding of the present invention.

[0055] Referring first of all to FIG. 1a, the bundle of rays constituting
illuminating light beam 12 uses the full aperture of illumination optics
10; i.e., passes through the entire area of entrance pupil 14 of
illumination optics 10, which is typical in conventional point-by-point
confocal scanning. In FIG. 1a, the optical axis of illumination optics 10
is denoted by O1. In the case depicted in FIG. 1a, illuminating light
beam 12 is aligned parallel to optical axis O1 of illumination optics 10.
Thus, illumination optics 10 produces a focused light distribution in the
form of illumination focus 16, which has a greater extent along optical
axis O1 than transverse to optical axis O1.

[0056] In the following explanations, reference is made to a coordinate
system whose x-axis is oriented horizontally in the plane of the paper
and whose z-axis is oriented vertically in the plane of the paper, while
the y-axis points out of the plane of the paper. With this definition,
entrance pupil 14 is parallel to the x-y plane, while optical axis O1
extends parallel to the z-axis.

[0057]FIG. 1b shows illumination focus 16 in a top view, looking along
optical axis O1. In this conventional set-up, illumination focus 16 is
circular in top view.

[0058] FIG. 2 illustrates how the position of illumination focus 16
changes when illuminating light beam 12 is tilted in entrance pupil 14 of
illumination optics 10. In the case shown in FIG. 2a, it is assumed that
the central ray of the bundle of rays constituting illuminating light
beam 12 is tilted in a plane of incidence which is parallel to the x-z
plane. It is also assumed that illuminating light beam 12 still passes
through the entire area of entrance pupil 14; i.e., illuminates the
entire entrance pupil 14.

[0059] Illuminating light beam 12 is tilted in the plane of incidence such
that it changes its direction of incidence relative to optical axis O1.
This change in the direction of incidence is translated by illumination
optics 10 into a movement of illumination focus 16 transverse to optical
axis O1. In the case shown in FIG. 2a, this movement is along the x-axis.
Since illuminating light beam 12 still passes through the entire area of
entrance pupil 14, illumination focus 16 remains oriented such that its
longitudinal extent is parallel to optical axis O1. This can also be seen
in the top views of FIG. 2b, in which the illumination focus 16 is still
circular.

[0060]FIG. 3a illustrates the underillumination of entrance pupil 14 of
illumination optics 10 according to the present invention. As can be seen
in FIG. 3a, illuminating light beam 12 passes through only a portion of
entrance pupil 14, said portion being located off-center; i.e., offset
from the center of the pupil transversely to optical axis O1. This
off-center position of illuminating light beam 12 in entrance pupil 14
results in illumination focus 16 being inclined relative to optical axis
O1. This can also be seen in the top view of FIG. 3b, in which
illumination focus 16 is no longer circular, but rather is longer in the
direction of the x-axis than in the direction of the y-axis. Moreover,
underillumination of entrance pupil 14 produces a widening of
illumination focus 16; i.e., illumination focus 16 is overall larger than
when illuminating the full entrance aperture 14.

[0061]FIG. 4a shows the case where an illuminating light beam 12 that
under-illuminates entrance pupil 14 at an off-center position is tilted.
In the case shown in FIG. 4a, it is assumed that the central ray of the
bundle of rays constituting illuminating light beam 12 is tilted in a
plane of incidence which is parallel to optical axis O1 and parallel to
the y-z plane. This means that in FIG. 4a, illuminating light beam 12 is
tilted out of and into the plane of the paper. This tilting of
illuminating light beam 12 is translated by illumination optics 10 into a
corresponding movement of the inclined illumination focus 16 along the
y-axis. Accordingly, in FIG. 4a, illumination focus 16 is moved out of
and into the plane of the paper. This is also illustrated in FIG. 4b.

[0062] The motion sequence of illumination focus 16 shown in FIG. 4b
illustrates how a light sheet 18 is generated within a scanning period by
moving illumination focus 16. The aforementioned scanning period is the
period of time within which illuminating light beam 12 performs a
complete tilting movement. This scanning period is shorter than a
detection period with which a photodetector (not shown in FIGS. 4a and
4b) operates to generate an image; i.e., to capture the moving
illumination focus 16. This means that the photodetector captures
illumination focus 16 in a temporally and, therefore, spatially
unresolved manner. Rather, it detects a continuous light distribution in
the form of light sheet 18.

[0063] FIG. 5 shows in schematic form a scanning confocal microscope 100
as a first exemplary embodiment. Scanning microscope 100 is configured to
operate according to the illumination principle delineated in FIGS. 3 and
4. In this connection, it should be noted that components of scanning
microscope 100 which are not essential to the understanding of the
subject matter of the present invention are omitted in the view of FIG.
5.

[0064] Scanning microscope 100 is configured as a confocal fluorescence
microscope having a laser light source 20 which emits illuminating light
beam 12. The wavelength of illuminating light beam 12 is selected such
that the fluorescent dyes employed in the microscopy technique used are
excited by illuminating light beam 12 to emit fluorescent radiation.

[0065] Illuminating light beam 12 strikes a mirror 22 which directs it to
a galvanometer minor system 24, which is shown purely schematically in
FIG. 5. Galvanometer mirror system 24 serves to deflect illuminating
light beam 12 in such a way that it performs the scanning motion
illustrated in FIG. 4a. To this end, galvanometer minor system 24 is
correspondingly moved by a mirror actuator (not explicitly shown).
Subsequently, illuminating light beam 12 passes through a scanning lens
26 and a tube lens 28 and finally strikes illumination optics 10.

[0066] The optical axis along which illuminating light beam 12 is incident
on illumination optics 10 is designated O2 in FIG. 5. Optical axis O2 is
parallelly offset from optical axis O1 of illumination optics 10, which
extends centrally through entrance pupil 14. Moreover, the illuminating
light beam 12 entering illumination optics 10 does not fully illuminate
entrance pupil 14. This means that illuminating light beam 12 passes
through only a portion of entrance pupil 14, and thus, does not use the
full aperture of illumination optics 10. This underillumination of
entrance pupil 14 is achieved by an aperture 29 which suitably restricts
the diameter of illuminating light beam 12 emitted by laser light source
20. In the exemplary embodiment shown in FIG. 5, aperture 29 is
positioned upstream of mirror 22. However, it may also be disposed at the
other location [sic] in the path of illuminating light beam 12. The
opening of aperture 29 is variably adjustable, whereby the
cross-sectional area of the illuminating light beam 12 incident on
entrance pupil 14 of illumination optics 10, and thus the
underillumination of entrance pupil 14, can be varied as desired.

[0067] In order to be able to variably adjust the off-center portion of
entrance pupil 14 that is traversed by illuminating light beam 12,
illumination optics 10 are mounted so as to be movable along an
adjustment direction, which is denoted in FIG. 5 by double-headed arrow
A. Adjustment direction A is perpendicular to optical axis O1 of
illumination optics 10.

[0068] A first actuator 30 coupled to illumination optics 10 serves to
move illumination optics 10 along adjustment direction A. Actuator 30 is
controlled by a control unit 32.

[0069] Components 24, 26, 28, 30 and 32 shown in FIG. 5 form part of a
scanning device, generally designated 33, which serves to tilt
illuminating light beam 12 in entrance pupil 14 of illumination optics 10
in the manner illustrated in FIGS. 4a and 4b. Scanning device 33
cooperates with illumination optics 10 to generate light sheet 18, which
illuminates a target region to be imaged of an object 36 located on an
microscope slide 34.

[0070] Scanning microscope 100 further has an observation objective 38
which is aligned with respect to sample 36 such that its such that its
optical axis, which is designated O3 in FIG. 5, is perpendicular to light
sheet 18. Since light sheet 18 is inclined relative to optical axis O1 of
illumination optics 10, optical axis O3 of observation objective 38 and
optical axis O1 of illumination optics 10 form an acute angle ?, which is
less than 90 degrees. In the exemplary embodiment shown in FIG. 5, angle
?is about 60 degrees.

[0071] The target region of object 36 that is illuminated with light sheet
18 is imaged by observation objective 38 through a tube lens 40 onto a
photodetector 42. In this process, optical axis O3 of observation
objective 38 is perpendicular to a light-receiving surface 44 of
photodetector 42. Observation objective 38, tube lens 40 and
photodetector 42 form a detection unit 46, which can be tilted by a
second actuator 47 to align optical axis O3 perpendicular to light sheet
16 [sic. 18], as indicated in FIG. 5 by double-headed arrow B. Second
actuator 47 is also controlled by control unit 32.

[0072] Control unit 32 controls the two actuators 30 and 47 such that when
illumination optics 10 is moved along adjustment direction A, detection
unit 46 including observation objective 38 is adjusted in position by
tilting it in direction B so as to maintain image-capturing lens [sic.
observation objective] 38 oriented with its optical axis O3 perpendicular
to the positionally variable light sheet 18. Thus, the movement of
illumination optics 10 along adjustment direction A and the tilting of
detection unit 46 in tilting direction B are synchronized by control unit
32 so as to at all times maintain the perpendicular alignment of
observation objective 38 with respect to light sheet 18.

[0073] The schematic view of FIG. 6 illustrates how in the exemplary
embodiment shown in FIG. 5, movement of illumination optics 10
perpendicular to optical axis O1 varies the inclination of illumination
focus 16. As shown in the left sub-figure of FIG. 6, elongated
illumination focus 16 is aligned along optical axis O1 when illumination
beam 12 is incident centrally on entrance pupil 14 of illumination optics
10. When illumination optics 10 is moved perpendicularly to its optical
axis O1 such that illuminating light beam 12 shifts from the center
toward the edge of the pupil, then illumination focus 16 is increasingly
inclined relative to optical axis O1. This illustrates how the
orientation of light sheet 18 generated by illumination focus 16 can be
varied by moving illumination optics 10.

[0075]FIG. 8 shows a scanning microscope 200 as a second exemplary
embodiment. The components of scanning microscope 200 which correspond
with those of scanning microscope 100 shown in FIG. 5 have the same
reference numerals as in FIG. 5 and will not be described again below.

[0076] The exemplary embodiment shown in FIG. 8 differs from that of FIG.
5 in that, in place of observation optics [sic. illumination optics] 10,
a transparent plane-parallel plate 50, for example, a glass plate, is
mounted so as to be tiltable in a tilting direction C to adjust the
off-center entry of illuminating light beam 12 into entrance pupil 14 of
illumination optics 10. Plane-parallel plate 50 is disposed in the path
of illuminating light beam 12 between tube lens 28 and entrance pupil 14
and is aligned at an angle to optical axis O1 of illumination optics 10.
Because of this, illuminating light beam 12 undergoes a parallel
displacement as it passes through plane-parallel plate 50, causing it to
enter entrance pupil 14 of illumination optics 10 at an off-center
position.

[0077] The parallel displacement of illuminating light beam 12 can be
varied. To this end, plane-parallel plate 50, which is mounted so as to
be tiltable about a pivot axle 51 extending perpendicularly to the plane
of FIG. 8, is tilted by the actuator 30 in tilting direction C.

[0078]FIG. 9 shows a scanning microscope 300 as a third exemplary
embodiment. Scanning microscope 300 is modified from microscope 200 shown
in FIG. 8 in that a transparent wedge-shaped plate 60, for example, a
glass wedge is used in place of plane-parallel plate 50. Wedge-shaped
plate 60 also causes a lateral displacement of illuminating light beam 12
when it passes through wedge-shaped plate 60. In this exemplary
embodiment, actuator 30 does not move wedge-shaped plate 60
perpendicularly to optical axis O1 of illumination optics 10, but along
an adjustment direction D which is at an angle to optical axis O1.

[0079]FIG. 10 shows a scanning microscope 400 as a fourth exemplary
embodiment. This exemplary embodiment differs from that shown in FIG. 8
in that an aperture plate 52, which is mounted so as to be movable along
adjustment direction A, is provided in place of plane-parallel plate 50
to achieve off-center entry of illuminating light beam 12 into entrance
pupil 12. Aperture plate 52 has a variable aperture opening 54 which
restricts the diameter of illuminating light beam 12 as it passes
therethrough so as to obtain the desired underillumination of entrance
pupil 14. Since aperture opening 54 can be adjusted as desired, the
aperture element 29 provided in the aforedescribed exemplary embodiments
can be omitted in the exemplary embodiment shown in FIG. 10.

[0080]FIG. 11 once again illustrates the function of aperture plate 52.
The portion of illuminating light beam 12 that passes through aperture
opening 54 is displaced from the center of the pupil by actuator 30
moving aperture plate 52 perpendicularly to optical axis O1 of
illumination optics 10. As the portion of illuminating light beam 12 that
is allowed to pass through shifts from the center toward the edge of the
pupil, illumination focus 16 is increasingly inclined with respect to
optical axis O1.

[0081] In FIG. 11, aperture plate 54 [sic. 52] is shown at some distance
from entrance pupil 14 to simplify the representation. However, it should
be noted that it is preferred for aperture plate 54 [sic. 52] to be
disposed as close as possible to entrance pupil 14 in order not to
negatively affect the tilting motion of illuminating light beam 12 in
entrance pupil 14, which serves to move illumination focus 16.

[0082]FIG. 12 shows a scanning microscope 500 as a fifth exemplary
embodiment. Scanning microscope 500 is modified from the exemplary
embodiment shown in FIG. 5 in that it is designed to achieve a spatial
resolution beyond the diffraction limit using the generally known STED
method. In this connection, it should be noted that this modification is
not only possible for the exemplary embodiment shown in FIG. 5, but also
for all other exemplary embodiments.

[0083] Scanning microscope 500 includes an excitation light source 61,
which emits an excitation beam 62 whose wavelength is selected such that
the fluorescent dyes used are excited to emit fluorescent radiation.
Scanning microscope 500 further includes a depletion light source 64,
which emits a depletion beam 66 which is superimposed on excitation beam
62 in a manner described further below, and whose wavelength is selected
such that the fluorescent dyes illuminated by excitation beam 62 are
depleted by stimulated emission, and thus, as it were, switched off.
Depletion beam 66 emitted by depletion light source 64 passes through a
phase plate 68 which serves to achieve the desired intensity profile for
depletion beam 66.

[0084] Excitation beam 62 passes through an aperture 70 and strikes a
minor 72 which reflects excitation beam 62 toward a beam splitter 74.
Beam splitter 74 is adapted to let excitation beam 62 through and to
reflect the depletion beam 66 passing through phase plate 68 and a
further aperture 76. In this manner, excitation beam 62 and depletion
beam 66, whose intensity profile is influenced by phase plate 68, are
superimposed on each other. The two superimposed beams 62 and 66 then
form illuminating light beam 12.

[0085] FIGS. 13a and 13b illustrate how the superposition of excitation
beam 62 and depletion beam 66 leads to an increase in the resolving power
of the microscopy method used.

[0086] Since, in this case, illuminating light beam 12 is formed by
superposition of excitation beam 62 and depletion beam 66, illumination
optics 10 produce an excitation focus 80 and a depletion focus 82, which
are superimposed to form an illumination focus, which is denoted by 84 in
FIGS. 13a and 13b. Excitation focus 80 shown in FIGS. 13a and 13b
corresponds in shape to illumination focus 16 shown in FIGS. 3 and 4.

[0087] In FIG. 13b, excitation focus 80, depletion focus 82, and the
resulting illumination focus 84 are shown in a sectional view parallel to
the x-z plane. As can be seen in this view, depletion focus 82 has a
spatial light intensity distribution where the light intensity is zero in
the plane in which the illumination focus 84 composed of excitation focus
80 and depletion focus 82 is moved, and exhibits a maximum on both sides
of said plane. The aforementioned plane, designated 86 in FIG. 13b, lies
parallel to a plane which contains the y-axis and whose line of
intersection with the x-z plane forms an angle with the x-axis. This
angle is dependent on the maximum acceptance angle of the illumination
optics 10 used and on the degree to which illuminating light beam 12 is
widened and displaced from the center.

[0088] Since excitation focus 80 is superimposed with depletion focus 82
above and below this plane of motion 86, the excitation effect of
excitation focus 80 is reduced from above and below said plane of motion
86. The portion of excitation focus that is effective in terms of
excitation is shown in hatched shading in FIG. 13b.

[0089] In FIG. 13b, the direction in which excitation focus 62 is, as it
were, constricted is indicated by a line 88. This direction of
constriction 88 is perpendicular to plane of motion 86. The superposition
of excitation focus 80 and depletion focus 82, as it were, makes the
excitation-producing light sheet thinner, thereby increasing the spatial
resolution.